Research Project :: Botulinum Research Center ::

Botulinum Toxin : A Perfect Model for Protein Chemistry :

In Silico structural & functional study of BoNT/A LC

We have explored the folding dynamics of the BoNT endopeptidase using BoNT serotype A Light Chain (BoNT/A LC) as a model system.
We have utilized urea denaturation assays and Molecular Dynamics (MD) simulations to unmask the folding mechanism.
A highly unusual folding pattern has emerged, which does not show a typical folding co-operativity but rather, it follows
a unique three-step denaturation process with two intermediate states, i.e. N <=>I1 <=>I2<=>U. This finding agreed with results
from MD simulations which enabled us to resolve the structural underpinning of BoNT in the native state (N), intermediate state
(I1 and I2), and unfolded state (U). Except for the intermediate state I2, all the states of BoNT/A retained full enzymatic activity
for the substrate SNAP-25, including the unfolded state U stable in 7 M urea solution. Direct observation of the functionally active
intermediate states of BoNT defines a unique folding pattern and specifies the crucial role played by partially unfolded intermediate
conformations. Our results stress the importance of the structural heterogeneity and conformational flexibility in the toxin's mechanism
of intracellular survival and action.

We are also examining the characteristics of native and PRIME (Pre Imminent Molten Globule Enzyme) conformations and mechanism of
enzyme-substrate binding, utilizing MD simulation.

Basic Research :

Structure & Composition : The seven well known serotypes of BoNTs are produced in for form of varying sizes of complexes, each consisting of BoNT and group of
neurotoxin associated proteins (NAPs). There is a need to understand the interplay between BoNT and NAPs in the production and release
of these complexes from Clostridium botulinum, and in the functioning of the BoNT under physiological and therapeutic conditions.
These proteins also provide a rich source of data to understand their evolution and the molecular features of evolution itself.

Molecular mechanism of BoNTs : BoNTs cause muscle flaccid paralysis by blocking the neurotransmitter release at the neuromuscular junctions.
The proposed mode of action of BoNTs involves the specific binding of BoNTs with the receptor on the nerve cells; the endocytosis of the toxin
into nerve cell and translocation of the toxins from endosome to cytosome of the nerve cells, and the cytotoxicity of BoNTs by being a selective
endopeptidase targeting SNARE proteins, which are critical for neurotransmitter release. We are working on all three proposed steps of the mode of
action of BoNTs, receptor binding, membrane translocation, and endopeptidase activity, to elucidate the molecular basis of BoNTs.

Protein Purification & Recombinant Protein Expression :
We routinely isolate and purify all the seven serotypes of BoNT from bacterial cultures.
We have developed the molecular biology experiences to clone, express, and purify the recombinant light and heavy chains of BoNTs, the SNARE proteins,
and the mutants of those proteins.

Host Response :
Current knowledge is that BoNT blocks neurotransmitter release by intracellular endopeptidase activity. However, given the fact that
the toxin can survive for months inside neuronal cells, it is likely to influence the genetic and metabolic activity of cells. The Center scientists
are carrying out research with microarray, cytokine response, and cellular apoptosis to delineate host response to BoNT exposure.

Applied Research :

Development of biosensors and detection systems : BoNT is the most toxic substance known to man. The only accepted detection method for BoNTs
is mouse bioassay,which need to take four days to complete, and cannot meet clinical diagnostics need. We are working on development of biosensors
to detect BoNT with the sensitivity similar or better than mouse bioassay, and a detection time of less than one hour.

Development of Vaccines and Antidotes against BoNTs : We are developing an oral vaccine against botulism. By employing the non-toxic components of
BoNT complex, we are trying to develop delivery system for oral vaccines. We are also working on development the antidotes against the endopeptidase
activity of BoNTs, which will help develop effective antidotes against botulism, even after patients have developed the syndrome.

Forensic Analysis : Clostridium botulinum has many strains producing seven serotypes of BoNT and over two dozen subtypes. Even if the same serotype and
subtype of BoNT is obtained its bacterial source may differ. The Center in collaboration with CDC has started a groundbreaking work in genomics for
identifying different isolates of the same strain of C. botulinum, thus allowing forensic analysis of the pathogenic agent.

Medical Research :

Drug formulation : The challenge in the development of protein-based therapeutic agents is the stability of protein and the alternative delivery route
rather than injection. We are working on both these fronts.

BoNT-based Drug Delivery Vehicles : BoNT with its extreme specificity for neuronal cells is now being targeted for designing drug delivery vehicle in
combination with nanoparticles and liposomes. Proof of concept research has already been carried out at the Center, and currently proposals are
being prepared to obtain funding to develop drug delivery vehicles against several neuronal and brain diseases, including amyotrophic lateral sclerosis
disease.

Publication - Differential Endopeptidase Activity of Different Forms of Type A Botulinum Neurotoxin: A Unique Relationship Between the Size of the Substrate and Activity of the Enzyme. Toxicon (Accepted)